Acute myeloid leukemia (AML) is the leading cause of adult acute leukemia and accounts for ˜80% of all adult leukemia (Menzin et al., 2002). Despite the extensive research done to develop more effective ways of targeting the disease, AML is associated with low long-term survival; only ˜5% of elderly patients and ˜30% younger patients with AML manage to survive for 5 years or longer (Ungewickell and Medeiros, 2012; Hoand et al., 2012). Conventional chemotherapy can effectively achieve initial remission of the disease in >70% of the treated AML patients (Ungewickell and Medeiros, 2012). However, due to the highly heterogeneous nature of the disease, ˜30% of AML patients do not respond to chemotherapy (Ungewickell and Medeiros, 2012; Bucisano et al., 2012). Furthermore, chemotherapy fails to achieve complete clearance of the disease in most patients, and more than 70% of patients in remission suffer from relapsing AML within 2 years after the initial treatment (Ungewickell and Medeiros, 2012; Bucisano et al., 2012). There is no standard treatment regime for patients with relapsing AML, which is associated with poor prognosis (Ferrara et al., 2004). Relapsing AML is caused by a phenomenon called minimal residual disease (MRD), which is mediated by an AML cell population with resistance to chemotherapy (Garces-Eisele, 2012; Lin and Levy, 2012). It is known that MRD is largely contributed by leukemic stem cell (LSC) population, as it has the ability to withstand harsh environment and conditions, such as chemotherapy (Ishikawa et al., 2010; Kadowaki and Kitawaki, 2011; Vaz et al., 2013). Therefore, development of treatments to target AML-LSC and MRD to achieve relapse-free clearance of the disease has been an active area of research.
Allogeneic hematopoetic stem cell transplantation (allo-HSCT) is a potential curative treatment for AML patients and is associated with higher disease-free survival rates than conventional chemotherapy (Alatrash and Molldrem, 2009). Donor-derived T cell mediated anti-leukemic effects contribute to the increased survival in patients, as T cell depleted grafts result in higher relapse rates (Alatrash and Molldrem, 2009). However, the use of allo-HSCT in the clinic is limited by a shortage of suitable donors, the toxicity of the treatment, and other associated complications (Alatrash and Molldrem, 2009; Shlomchik, 2007). Potent immune responses can be induced on normal tissues, resulting in tissue damage and, possibly, in death of the patients in severe cases (Alatrash and Molldrem, 2009; Shlomchik, 2007) thus posing a major obstacle that limits the use of allogenic cellular therapies.
Since the early work on utilizing T cell immunotherapy to treat melanoma patients, significant progress has been made in adoptive T cell therapy for other cancers, which further supports the potential use of cellular therapies to achieve relapse-free AML clearance (Rosenberg et al., 1988). Antigens that are upregulated in leukemic cells, leukemia associated antigens (LAA), have been identified, and the anti-leukemic effect of LAA-specific T cells has been demonstrated in vitro and in animal models (Vaz et al, 2013; Teague and Kline, 2013). However, the use of LAA-specific T cells is hampered by difficulties in isolation and expansion of these cells (Kochenderfer et al., 2010; Johnson et al., 2009; Parkhurst et al., 2011; Robbins et al., 2011). Furthermore, even though many LAAs are over expressed in AML, expression of the antigens in other tissues such as thymus prevents development of mature T cells with receptors that have high avidity towards LAAs due to thymic selection of T cell specificity (Teague and Kline, 2013). Alternatively, attempts have been made to use transgenic CD8+ T cells expressing transgenic TCRs or chimeric Ag receptor against LAAs, such as Wilms' tumour antigen or Lewis Y, respectively (Peinert et al., 2010; Xue et al., 2010). These T cells have a significantly increased ability to bind to LAAs and show excellent anti-tumour activity (Kochenderfer et al., 2010; Johnson et al., 2009; Parkhurst et al., 2011; Robbins et al., 2011). However, the potential side-effects associated with gene therapy, together with complicated and long procedures, imposes limitations on using these strategies to treat AML. In addition, injecting supra-physiological numbers of genetically engineered T cells can lead to severe adverse events, including death. Thus, the development of new cellular immunotherapies with potent effects on a broad range of cancers without the requirement of identifying LAAs may revolutionize leukemia immunotherapy.
Double negative T cells (DN T cells or DNTs) are mature peripheral T lymphocytes that express the CD3-TCR complex but do not express CD4, CD8, or NKT cell markers αGalCer-loaded CD1d and Jα24-Vα14; they represent 1˜3% of peripheral blood mononuclear cells (PBMC) in humans (Zhang et al., 2000). Protocols for expanding DNTs from AML patients during chemotherapy-induced complete remission have been described and AML patient DNTs have been shown to have significant anti-leukemic activity against the primary AML cells obtained from the same patient in vitro (Young et al., 2003; Merims et al., 2011).
Previously, DNTs have been shown to induce the killing of an allogeneic AML cell-line in a dose-dependent manner through the perforin-granzyme dependent pathway (Merims et al., 2011). In animal models, it has also been shown that unlike conventional CD4+ or CD8+ T cells, infusion of allogeneic mouse DNTs may confer immune inhibitory function (Zhang et al., 2000; Young et al., 2003; He et al., 2007). However, the activity of DNTs with respect to patient primary leukemic cells had not been studied in vivo.